An evaluation of inflammatory and endothelial dysfunction markers as determinants of peripheral arterial disease in those with diabetes mellitus

The most serious long-term effects of diabetes is peripheral artery disease (PAD) which increases the chance of developing diabetic foot ulcers, gangrene and even lower limb amputation. The clinical manifestations of PAD which are typically not revealed until symptoms like intermittent claudication, rest pain and ischemic gangrene develop, are not present in majority of diabetes mellitus patients with PAD due to diabetic peripheral neuropathy. Therefore, current study is aimed to evaluate the inflammatory and endothelial dysfunction markers with their correlation to biomarkers that can help for in-time diagnosis and efficient prognosis of developing diabetes-associated PAD. Enzyme-linked immunosorbent assay was used to evaluate the interlukin-6, interlukin-8, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) in PAD with diabetes group, diabetic group and healthy individual group while biomarkers were measured by kit method. It was observed that serum IL-6, IL-8, ICAM and VCAM levels in type II diabetes mellitus (T2DM) with PAD patients were increased significantly (85.93, 597.08, 94.80 and 80.66) as compared to T2DM patients (59.52, 231.34, 56.88 and 50.19) and healthy individuals (4.81, 16.93, 5.55 and 5.16). The overall means for the parameters, IL-6, IL-8, ICAM, VCAM, urea, S/creatinine, CK-MB, AST, ALT, cholesterol, triglyceride, HDL, LDL, PT, aPTT, INR, HbA1C, and CRP within all groups were significantly (P < 0.05) different from each other. Therefore, it was concluded that the change in IL-6, IL-8, ICAM and VCAM can serve as an accurate diagnostic indicator and successful treatment.


Sample collection and preparation
10 mL of venous blood samples from DM, DM plus PAD, and healthy individuals were collected who visited the Mayo Teaching Hospital of King Edward Medical University (KEMU), Lahore, according to WHO guidelines following the consent of the patients involved in the research 43 .Blood samples were divided into two experimental groups and one control group.Diabetic blood samples (D-S1 and D-S2) were included in group 1 while blood samples from diabetics with PAD (DP-S1 and DP-S2) were included in group 2, and blood samples from healthy individuals were labeled as a control.Subsequently, subsamples were prepared by drawing 3 mL of blood from each tube into a gel vial with yellow cap for biochemical profiling, 2 mL in a citrated vial with blue cap for coagulation profiling, and 5 mL in a sterile vial with red cap in ethylenediaminetetraacetic acid (EDTA) for HbA1c analysis 44,45 .

Interleukin-6 (IL-6)
Interleukin-6 is multifunctional cytokine that regulates immune responses, acute phase reactions and hematopoiesis and plays an important role in host defense mechanisms.Interleukin-6 was measured in serum samples via human IL-6 ELISA kit of Thermo Fischer Scientific.The serum was collected after centrifugation of blood at 4000 xg for 10 min.Lyophilized human IL-6 standard was reconstituted with sterile distilled water.Label seven sterile tubes, one for each standard marked as S1, S2, S3, S4, S5, S6 and S7.Prepared 1:2 serial dilutions for standard curve as follows; Take 225 μL of assay buffer (1x) into each tube.Pipette 225 μL of reconstituted standard (200 pg/ mL) into the first tube S1 and mix it gently.Take 225 μL of this dilution into 2nd tube S2 and mix thoroughly.Repeat serial dilutions 5 more times S33, S4, S5, S6 and S7.Lyophilized controls were reconstituted with distilled water.Pipette 100 μL of each standard dilution in the microwells.
Serum samples were diluted 1:2 (50 μL sample + 50 μL 1 × assay buffer) as add 50 μL assay buffer 1 × in samples well 50 μL serum, 50 μL high and low control and 100 μL in blank.An anti-human IL-6 antibody is adsorbed onto microwells of ELISA plate.Human IL-6 present in the serum or standard binds to antibodies adsorbed to the microwells.A 50 μL biotin-conjugated anti-human IL-6 antibody was added to all wells, incubated for 2 h at room temperature (18-25 °C) and bound to human IL-6 captured by the first antibody.Microwells were emptied and washed 4 times with wash buffer.A 100 μL of Streptavidin-HRP were added to all wells and incubated for 1 h at room temperature (18-25 °C).Microwells were emptied and washed 4 times with wash buffer.A 100 μL of TMB substrate solution was added to all wells and incubated at room temperature (18-25 °C) for 10 min.A 100 μL of stop solution was added to all wells and color density was measured at 450 nm in ELISA microplate reader 46 .

Interleukin-8 (IL-8)
Interleukin-8/Neutrophil-Actvating Peptide-1 selectively stimulates the ability of neutrophils and T-lymphocytes to invade injured or inflamed tissue.Interleukin-8 was measured in serum samples via human IL-8 ELISA kit of Thermo Fischer Scientific.The serum was collected after centrifugation of blood at 4000 xg for 10 min.Lyophilized human IL-8 standard was reconstituted with sterile distilled water.Label seven sterile tubes, one for each standard marked as S1, S2, S3, S4, S5, S6 and S7.Prepare 1:2 serial dilutions for standard curve as follows; Take 225 μL of assay buffer (1x) into each tube.Pipette out 225 μL of reconstituted standard (200 pg/ mL) into the first tube S1 and mix it gently.Take 225 μL of this dilution into 2nd tube S2 and mix thoroughly.Repeat serial dilutions 5 more times S3, S4, S5, S6 and S7.Lyophilized controls were reconstituted with distilled water.Pipette 100 μL of each standard dilution in the microwells.
Serum samples were diluted 1:2 (50 μL sample + 50 μL 1 × assay buffer) as add 50 μL assay buffer 1 × in samples well, 50 μL serum, 50 μL high and low control and 100 μL in blank.An anti-human IL-8 antibody is adsorbed onto microwells of ELISA plate.Human IL-8 present in the serum or standard binds to antibodies adsorbed to the microwells.A 50 μL biotin-conjugated anti-human IL-8 antibody was added to all wells, incubated for 2 h at room temperature (18-25 °C) and bound to human IL-8 captured by the first antibody.Microwells were emptied and washed 3 times with wash buffer.A 100 μL of Streptavidin-HRP were added to all wells and incubated for 1 h at room temperature (18-25 °C).Microwells were emptied and washed 3 times with wash buffer.A 100 μL of TMB substrate solution was added to all wells and incubated at room temperature (18-25 °C) for 10 min.A 100 μL of stop solution was added to all wells and color density was measured at 450 nm in ELISA microplate reader 46 .

Intercellular adhesion molecule-I (ICAM-I)
Intercellular Adhesion molecule-I (ICAM-I) is a member of the immunoglobulin supergene family and functions as a ligand for the lymphocyte function-associated antigen-1.ICAM-I was measured in serum samples via human ICAM-I ELISA kit of Thermo Fischer Scientific.The serum was collected after centrifugation of blood at 4000 xg for 10 min.Lyophilized human ICAM-I standard was reconstituted with sterile distilled water.Label four sterile tubes, one for each standard marked as S1, S2, S3 and S4.Prepare 1:2 serial dilutions for standard curve as follows; Take 225 μL of sample diluent into each tube.Pipette out 225 μL of reconstituted standard (1 = 100 ng/mL) into the first tube S1 and mix it gently.Take 225 μL of this dilution into 2nd tube S2 and mix thoroughly.Repeat serial dilutions 2 more times S3 and S4.Lyophilized controls were reconstituted with distilled water.Pipette 100 μL of each standard dilution in the microwells.
Serum samples were diluted 1:10 (10 μL sample + 90 μL sample diluent) as add 90 μL sample diluent in samples well with 10 μL serum, 10 μL high and low control and 100 μL in blank.An anti-human ICAM-I antibody is adsorbed onto microwells of ELISA plate.Human ICAM-I present in the serum or standard binds to antibodies adsorbed to the microwells.A 50 μL HRP-conjugated was added to all wells, incubated for 1 h at room temperature (18-25 °C) and bound to human ICAM-I captured by the first antibody.Microwells were emptied and washed 3 times with wash buffer.A 100 μL of Streptavidin-HRP were added to all wells and incubated for 1 h at room temperature (18-25 °C).Microwells were emptied and washed 3 times with wash buffer.A 100 μL of TMB substrate solution was added to all wells and incubated at room temperature (18-25 °C) for 10 min.A 100 μL of stop solution was added to all wells and color density was measured at 450 nm in ELISA microplate reader 47 .

Vascular cell adhesion molecule-I (VCAM-I)
Vascular cell adhesion molecule-I (VCAM-I) or CD106 is a member of the immunoglobulin supergene family.VCAM-I was measured in serum samples via human VCAM-I ELISA kit of Thermo Fischer Scientific.The serum was collected after centrifugation of blood at 4000 xg for 10 min.Lyophilized human VCAM-I standard was reconstituted with sterile distilled water.Label six sterile tubes, one for each standard marked as S1, S2, S3, S4, S5 and S6.Prepare 1:2 serial dilutions for standard curve as follows; Take 225 μL of sample diluent into each tube.Pipette out 225 μL of reconstituted standard (1 = 100 ng/ mL) into the first tube S1 and mix it gently.Take 225 μL of this dilution into 2nd tube S2 and mix thoroughly.Repeat serial dilutions 4 more times S3, S4, S5 and S6.Lyophilized controls were reconstituted with distilled water.Pipette 100 μL of each standard dilution in the microwells.
Serum samples were diluted 1:50 (10 μL sample + 490 μL assay buffer) as add 490 μL assay buffer in 10 μL serum, then take 100 μL prediluted sample in all sample wells, 10 μL high and low control and 100 μL in blank.An anti-human VCAM-I antibody is adsorbed onto microwells of ELISA plate.Human VCAM-I present in the serum or standard binds to antibodies adsorbed to the microwells.A 50 μL conjugate-mixture was added to all wells, incubated for 2 h at room temperature (18-25 °C) and bound to human VCAM-I captured by the first antibody.Microwells were emptied and washed 3 times with wash buffer.A 100 μL of TMB substrate solution was added to all wells and incubated at room temperature (18-25 °C) for 10 min.A 100 μL of stop solution was added to all wells and color density was measured at 450 nm in ELISA microplate reader 47 .

Estimation of glycan and glycosaminoglycan (HbA1c)
Measurement of hemoglobin-A1c was done on Beckman coulter system.Quantitative estimation of hemoglobin 1c concentration was performed in human whole blood.The HbA1c assay was carried out using the kit consisting of total hemoglobin reagent, HbA1c antibodies and HbA1c polyhepten and hemolyzing reagent 48 .K2-EDTA whole blood was pre-treated with hemolyzing reagent in ratio of 1:100 and mixed well to obtain complete hemolysis of blood.Tetradecylammonium bromide (TTAB) in hemolyzing reagent eliminates the white blood cells.The concentration of both HbA1c and total hemoglobin were determined.HbA1c reagent was used to measure hemoglobin A1c concentration by turbidimetric immunoinhibition method.Hemoglobin A1c The measurement of blood urea nitrogen (BUN) was carried out in serum samples of patients by Beckman Coulter AU analyzer.At first urease enzyme was added to the serum sample in order to hydrolyze urea in to ammonia and carbon dioxide.Subsequently, l-glutamate dehydrogenase catalyzed the conversion of ammonia and α-oxoglutarate to glutamate.In the whole process, the hydrolysis of a urea molecule was associated with the oxidation of two molecules of NADH.At the end the absorbance was determined at 340 nm which increases with the falling levels of NADH in the reaction mixture 54 .

Creatine kinase MB
The plasma levels of creatine kinase-MB (CK-MB) were estimated by the kit method.For obtaining plasma, the blood samples were centrifuged for 15 min at 500 xg at room temperature.After obtaining plasma, both reagents were added according to the manufacturer's instructions and the absorbance was recorded using a microplate reader 54 .

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
The levels of AST are measured in serum samples that were stored at 15-20 ℃ and analyzed by using Beckman Coulter AU analyzer.88 mmol/L of Tris buffer (pH 7.80), 900 U/L of lactate dehydrogenase (LDH), 260 nmol/L of l-aspartate, 600 U/L of malate dehydrogenase (MDH), 0.22 mmol/L of NADH and 12 mmol/L of α-oxoglutarate were used to measure AST.In this reaction, aspartate and α-oxoglutarate were catalyzed by AST to form oxaloacetate (OA) and l-glutamate.OA was reduced by MDH into l-malate in the presence of NADH which was converted in to NAD + .At the end of this reaction, the activity of AST was measured at 340 nm.The absorbance was found to decrease due to increase in the consumption of NADH indicating enhanced AST activity 52 .
ALT levels were also evaluated via Beckman Coulter AU analyzer with all ALT reagents prepared beforehand.At first amino group transfer reaction was catalyzed by ALT in which alanine and α-oxoglutarate were converted into glutamate and pyruvate.The reaction was further continued by the action of LDH on pyruvate in the presence of NADH to form lactate and NAD + .Afterwards, the absorbance was measured at 340 nm indicating the consumption of NADH and ALT activity 52 .

Total cholesterol
To evaluate the total cholesterol levels, isopropanol: NP40 v/v (9:1) was used to dissolve the sample.Subsequently, 10 µL/well of 100 U/mL of catalase solution and 40 µL/well of sample solution in a black 96-well microplate were added.The microplate was incubated for 15 min at 37 °C to remove any peroxidase in the reagent or sample.The second incubation for 15 min at 37 °C was carried out after the addition of 150 µL/well of 0.67 U/mL cholesterol esterase plus reagent A, containing 0.4 mM ADHP, 1.3 U/mL HRP, 0.1% Triton X-100, 5 mM cholic acid, 0.1 M potassium phosphate buffer (pH 7.4), into each well of the microplate.After the second incubation, the absorbance was measured at the excitation and emission wavelengths of 530 and 580 nm, respectively 55 .

Triglycerides
The GPO/POD method was used to determine triglyceride (TG) levels in the venous blood samples.Microbial lipases were added to the blood sample to initiate the hydrolysis of TGs yielding glycerol and free fatty acids (FFAs).Subsequently, glycerol was phosphorylated by glycerol kinase and glycerol-3-phosphate oxidase into glycerol-3-phosphate (G3P) and oxidizes G3P generating dihydroxyacetone phosphate (DAP) and hydrogen , respectively.The end product will be red quinoneimine produced by the reaction of H 2 O 2 with 4-aminoantipyrine and 4-chlorophenol.The absorbance measured will be proportional to the concentration of TGs in the sample evaluated 56 .

Low-and high-density lipoprotein
High-density lipoproteins (HDL) and low-density lipoproteins (LDL) were measured simultaneously by the homogeneous method 29 .For HDL, chylomicrons, LDL and very low-density lipoproteins (VLDL) were blocked by the formation of antigen-antibody complexes in the sample and HDL was measured by the enzymatic cholesterol.
In the case of LDL, two types of reagents were used to quantify LDL in the sample solution.At first, reagent 1, which contains cholesterol oxidase, cholesterol esterase, ascorbic acid, 4-amino-antipyrine, MES buffer (pH 6.3), peroxidase, and detergent 1, was added to each well with the sample solution.Two types of reactions occur which involve the solubilization of non-LDL proteins and degradation of released cholesterol by detergent 1 and enzymes, respectively.Secondly, reagent 2, containing detergent 2, MES buffer (pH 6.3), and N,N-bis-m-tolidinedisodium, was added to solubilize LDL which was later quantified 57 .

Statistical analysis
International Business Machine (IBM)-Statistical Package for Social Sciences (SPSS) version 23 was used for data analysis.Data was evaluated for overall means for various parameters using ANOVA.Significant difference of different parameters among various groups were evaluated using the repeated measures ANOVA (Dunnett's T3 Post Hoc Test).The results of renal function tests, cardiac function test, ALT, lipid profile, coagulation profile, HbA1c and C-reactive proteins were correlated by healthy individuals, participants with diabetes mellitus (DM) and those with DM plus peripheral arterial disease (PAD) using the Pearson's Correlation Coefficient (r value).

Ethical approval
The Research and Ethics Committee (Path/No.195/2021),Faculty of Pathology, King Edward Medical University, Lahore, Pakistan, approved the current study.

Evaluation of inflammatory and endothelial dysfunction markers and metabolic characteristics in patients with type 2 diabetes mellitus (T2DM) plus peripheral arterial disease (PAD), T2DM and healthy individuals
The ANOVA revealed that the overall means for the parameters, IL-6, IL-8, ICAM, VCAM, urea, S/creatinine, CK-MB, AST, ALT, cholesterol, triglyceride, HDL, LDL, PT, aPTT, INR, HbA1C, and CRP within all groups were significantly (P < 0.05) different from each other.
However, repeated measures ANOVA (Dunnett's T3 Post Hoc Test) between various groups revealed that the IL-6, IL-8, ICAM and VCAM in healthy persons were significantly lower than the all-other groups.The data presented in Table 1 shows the mean plus standard error mean of different groups.The results revealed that serum IL-6 level in type II diabetes mellitus (T2DM) with peripheral arterial disease (PAD) patients was increased

Diagnostic performance of inflammatory and endothelial dysfunction markers in PAD + T2DM
The ROC curves represent a correct diagnostic performance for all four inflammatory and endothelial dysfunction markers.The area under curve (AUC, s) for IL-6, IL-8, ICAM and VCAM were higher than 0.8 (Fig. 1).AUCs for two inflammatory markers and two endothelial dysfunction markers were highly statistically significant (p < 0.005, 0.001) (Table 7).

Discussion
T2DM increases the susceptibility to complex pro-thrombotic state where endothelial dysfunction, platelet hyperactivity, changes to coagulation cascade and chronic low-grade inflammation play significant role 58 .Inflammatory biomarkers play important role in atherosclerosis 59 .IL-6 is released by fibroblasts, endothelial cells, macrophages, B-lymphocytes and Th2 lymphocytes and is pivotal in developing inflammatory response in PAD with  60 .IL-6 has played important role in the genesis of atherosclerosis and releasing of IL-8 by endothelial cells and macrophages 61 .Numerous studies have examined the role of IL-6 in the pathophysiology of atherosclerosis particularly examining the relationship between the inflammatory process and peripheral artery disease (PAD).Numerous inflammatory molecules, sometimes referred to as acute-phase proteins, including CRP, fibrinogen synthesis, complement factor release, and serum amyloid A formation, are known to be stimulated by IL-6 in the liver 61,62 .IL-6 promotes the synthesis of MCP-1 and IL-8 by macrophages and endothelial cells, among other functions in the development and upkeep of atherosclerotic plaque.Additionally, IL-6 seems to be able to boost the synthesis of ICAM-1 by SMCs and increases the release of chemokines by the arterial wall's intimal cells.Furthermore, it facilitates leukocyte homing into atherosclerotic plaque.Lastly, IL-6 encourages SMCs to develop into foam cells.Research has demonstrated that IL-6 is a novel predictor of PAD and a valid marker for predicting the clinical course of the illness over a twelve-year span.Its function in PAD is actually well-defined, and evidence from community research indicates that it ought to be regarded as a predicting 63 .
Chronic consequences of diabetes mellitus (DM) include atherosclerosis in the arteries of the heart, brain and lower limbs.Leg ulcers and amputations can result from these issues that damage the lower limb arteries which is called PAD.Its burden is steadily increasing and contributes to diabetic's mortality 64 .Peripheral artery disease (PAD) typically coexists with the conditions like diabetes, cardiac disease and respiratory illness.It is linked to the gradual loss of limb; claudication and gangrene witch ultimately lowers quality of life 65 .As a result, evaluation of a number of inflammatory biomarkers for the early diagnosis of PAD, in order to lower the risk of mortality and enhance their quality of life, is crucial.
Greater the exposure of the biomarkers IL-6 and IL-8 has resulted in a collection of research data on the role of interleukins (ILs) in PAD, specifically in relation to glycoproteins acting as proinflammatory substances that influence blood vessel walls when atherosclerotic plaque development is initiated and how it develops.The acute phase protein known as C-reactive protein (CRP) originates from liver cells.The secretion of interlukin-6 by macrophages and T-cells promotes its release 60 .The release of IL-8 from monocytes and macrophages depends critically on stimulated inflammatory circumstances.It is intriguing to observe that polymorph nuclear Table 7.The diagnostic test performance for inflammatory and endothelial dysfunction markers in PAD + T2DM.AUC, area under curve; CI, confidence interval.AUC values for above all markers are significantly higher than 0.8 which is indicative of the accuracy of sensitivity analysis of a good model.The susceptibility to exhibit continuous atherosclerosis progression is one of the primary characteristics of peripheral artery disease.It is generally known that inflammation plays a crucial role in the emergence of atherosclerosis.However, there is no previously reported inflammatory biomarker for the identification of the risk of stratification or conclusive diagnosis of PAD 68 .T2DM has significant higher levels of ICAM and VCAM compared to controls, an adhesion molecule considered to play a role in atherosclerosis progression 69 .Our results agree with earlier study, ICAM and VCAM were all found to be positively linked with DM in a prior study by Bluher 70 as compared to hyperglycemic and impaired glucose tolerant patients.A more recent study reported a rise in VCAM blood concentration of PAD patients 71 .According to another recent report, VCAM is a marker that is pathogenically related to diabetic nephropathy and is predictive of it 68 .The results of our study revealed that serum ICAM level in type II diabetes mellitus (T2DM) with peripheral arterial disease (PAD) patients was increased significantly (94.80) as compared to T2DM patients (56.88) and healthy individuals (5.55).Serum VCAM level in type II diabetes mellitus (T2DM) with peripheral arterial disease (PAD) patients was increased significantly (80.66) as compared to T2DM patients (50.19) and healthy individuals (5.16).
To counteract the inflammation caused by other cytokines, IL-6 (as adipokine) levels gradually rise in DM, yet this can result in inflammation of vascular cells that leads to atherosclerosis 72 .Adipocytes in adipose tissue release IL-6 and IL-8 in obese individuals which induces insulin resistance particularly in hepatocytes and results in DM 73 .Due to the increased levels of cytokines and stress in DM, the levels of intercellular adhesion molecule (ICAM) and vascular cellular adhesion molecules (VCAM) are also increased.They are highly expressed in endothelial cells of arteries and trigger the release of new cytokines, which erodes the lining of the blood vessels 74 .IL-6, IL-8, ICAM and VCAM are rapidly rising in PAD causes the complications.So they induce endothelial abnormalities and harm the vascular system.
The levels of renal markers (BUN and serum creatinine) were also abnormally raised in DM plus PAD than in DM.BUN and creatinine are metabolic waste products that are normally excreted from kidneys, but under pathological conditions, they accumulate in the blood plasma 74 .In diabetics, the plasma level of creatinine drops due to decreased skeletal muscle volume; however, it rises in vascular disease indicating renal dysfunction and may increase or worsen atherosclerosis 75,76 .In critical limb ischemia, the level of BUN is also increased where it serves as a diabetic risk factor, a biomarker for hemodynamic changes, and an indicator of survival chances in patients 77 .In our study, the result of correlation coefficient of inflammatory markers (IL-6, IL-8) and endothelial dysfunction marker (ICAM, VCAM) with biochemical marker urea and creatinine of overall (IL-6, IL-8, ICAM, VCAM × urea, creatinine) had a statistical significantly positive correlation.
CRP is an indicator of cardiac dysfunction and functions as an acute phase reactant in blood plasma 78 .Its production is stimulated by an IL-6 mediated inflammatory signal in hepatocytes and adipose tissues; therefore, enhanced levels represent an inflammation or tissue injury 79,80 .As the level of CRP rises in blood so does the progression of atherosclerosis.It is because being acute phase reactant it activates platelets, complements system, initiates thrombus formation, and modifies innate immunity 81 .In our study, the result of correlation coefficient of inflammatory markers (IL-6, IL-8) and endothelial dysfunction marker (ICAM, VCAM) with biochemical marker AST, CKMB and CRP of overall (IL-6, IL-8, ICAM, VCAM × AST, CKMB and CRP) had a statistical significantly positive correlation.
Persistent glycemic control and prognosis of the T2DM therapy are critically associated with the levels of HbA1c, where, they represent the mean blood levels of glucose over a period of 3-6 months 82,83 .HbA1c levels are essentially elevated in T2DM as demonstrated by our results and agree with previous research 82 .Therefore, HbA1c can serve as an independent determinant for investigating diabetics that have the chance to develop PAD 84 .The association of HbA1c with PAD is due to increased glucose concentration over a long period of time thus activating platelets and protein kinase C pathway that induces pro-inflammatory condition leading to enhanced oxidation stress and endothelial dysfunction.Moreover, this consistent hyperglycemic condition also causes stiffness in the arteries 83 .In our study, the result of correlation coefficient of inflammatory markers (IL-6, IL-8) and endothelial dysfunction marker (ICAM, VCAM) with biochemical marker glycolytic hemoglobin (HbA1c) of overall (IL-6, IL-8, ICAM, VCAM × HbA1c) had a statistical significantly positive correlation.
Furthermore, the PT and aPTT (coagulation profile) level doesn't increase preferably in diabetics plus PAD as compared to the control group and diabetics group.Unlike, enhanced fibrinogen levels are reported in diabetic patients as compared to non-diabetics 85 .Furthermore, coagulation profile indicates the risk of atherothrombotic events, the main cause of the development of cardiovascular complications 86 .In our study, the result of correlation coefficient of inflammatory markers (IL-6, IL-8) and endothelial dysfunction marker (ICAM, VCAM) with coagulation markers (PT, aPTT) of overall (IL-6, IL-8, ICAM, VCAM × PT, APTT) had a statistical significantly positive correlation.
Liver function tests were performed to estimate the levels of AST and ALT.Both the enzyme levels were elevated in diabetics and diabetics plus PAD.The level of AST and ALT significantly increases among diabetics and non-diabetics (control).These results agree to those reported previously 87 .Contrarily, when diabetics develop PAD symptoms, marked elevation in the levels of AST and ALT occurs.Diabetes with PAD can also be linked with abnormalities in the lipid profile of patients.The levels of triglycerides are elevated while low levels of HDL were observed in our investigations which are in accordance with the literature reports 88 .In our study, the result of the correlation coefficient of inflammatory markers (IL- Although the level of all the tested parameters IL-6, IL-8, ICAM, and VCAM increased in DM plus PAD except and HDL levels, which are somehow, decreased from the control values.Any variation in markers of interest in healthy individuals, diabetics and diabetics plus PAD can be easily observed from Tables 1, 2, 3, 4, 5 and 6.A remarkable increase in IL-6, IL-8, ICAM and VCAM depicts that these analytes can be utilized as biomarkers in the diagnosis and prognosis of PAD associated with DM. The clinical manifestations of PAD include claudication and pain at rest which progresses to ulceration and gangrene in later stages.Nonetheless, diabetic neuropathy may also be a secondary cause of leg pain and functional impairment 89 .One tool for categorizing the clinical phases of symptomatic PAD is the Fontaine scale.Fontaine stages IIa and IIb have mild and moderate to severe impairment claudication, respectively; Fontaine stage-III patients have symptoms at rest; and Fontaine stage-IV patients have considerable tissue loss (ulcers or gangrene).Patients at Fontaine stage-I have PAD but are asymptomatic 90 .Significant elevation of inflammatory and endothelial dysfunction markers include IL-6, IL-8, ICAM and VCAM can diagnose PAD patients even at Fontaine stage-I.Along these lines, machine learning techniques could be helpful in combing many biomarkers with clinical and functional factors to provide prediction algorithms that are more accurate.This will result in more precise PAD risk assessment, earlier PAD diagnosis and more individualized medication or surgery regimens 91 .

Conclusion
In summary, diabetics can progressively develop PAD that damages their vascular system and increases the risk of cardiovascular disorders and lower limb amputations.Despite the incidence of PAD, poor therapeutic strategies have been used due to the lack of knowledge about the PAD clinical manifestations and limited markers for its early diagnosis, progression and prognostic evaluation in targeted manner.Keeping these problems in mind, we investigated a number of inflammatory and endothelial dysfunction markers along with biomarkers and found drastic alterations in the levels of inflammatory and endothelial dysfunction markers and some biomarkers in diabetics plus PAD as compared to diabetics only.These inflammatory and endothelial dysfunction markers include IL-6, IL-8, ICAM and VCAM.Among these biomarkers, marked increase in IL-6, IL-8, ICAM and VCAM were significantly higher in diabetics plus PAD as compared to DM and healthy individuals.In order to prevent the PAD in patients with DM, doctors may view these characteristics as useful diagnostic and prognostic criteria.The patients can be advised by clinicians to check their IL-6, IL-8, ICAM and VCAM from clinical pathology laboratory.
The current research also pointed out the interesting objectives particularly considering that vasoactive drugs commonly used in PAD treatment proved not to fulfill all the therapeutic targets.Further research is needed on additional inflammatory biomarkers in PAD to improve the database for their mechanisms and on novel effective therapies.

Figure 1 .
Figure 1.Receiver operating characteristics (ROC) for inflammatory and endothelial dysfunction markers.

Table 1 .
The Mean ± SEM of the different groups which were examined during the 2018-2022.Different superscript shows the statistical difference (P < 0.05) between the groups.

Table 2 .
The Pearson's correlation of Inflammatory and endothelial dysfunction markers of overall which were examined during the 2018-2022.**Correlation is significant at the 0.01 level (2-tailed), *Correlation is significant at the 0.05 level (2-tailed) and NS for non-significant.

Table 3 .
The Pearson's correlation of inflammatory marker (IL-6) with biochemical markers of overall which were examined during the 2018-2022.**Correlation is significant at the 0.01 level (2-tailed), *Correlation is significant at the 0.05 level (2-tailed) and NS for non-significant.

Table 4 .
The Pearson's correlation of inflammatory marker (IL-6) with biochemical markers of overall which were examined during the 2018-2022.**Correlation is significant at the 0.01 level (2-tailed), *Correlation is significant at the 0.05 level (2-tailed) and NS for non-significant.

Table 5 .
The Pearson's correlation of endothelial dysfunction marker (ICAM) with biochemical markers of overall which were examined during the 2018-2022.**Correlation is significant at the 0.01 level (2-tailed), *Correlation is significant at the 0.05 level (2-tailed) and NS for non-significant.

Table 6 .
The Pearson's correlation of endothelial dysfunction marker (VCAM) with biochemical markers of overall which were examined during the 2018-2022.**Correlation is significant at the 0.01 level (2-tailed), *Correlation is significant at the 0.05 level (2-tailed) and NS for non-significant.

Test result variables (s) AUC Std. Error a Asymptotic Sig. b Asymptotic 95% confidence interval Lower bound Upper bound
67ukocytes significantly produce IL-866.Significant associations between IL-18, P-selectin, VCAM and other inflammatory markers including IL-1, IL-6 and CRP as well as adhesion molecules like ICAM67.In our study, the results revealed that serum IL-6 level in type II diabetes mellitus (T2DM) with peripheral arterial disease (PAD) patients was increased significantly (85.93) as compared to T2DM patients (59.52) and healthy individuals (4.81).Serum IL-8 level in type II diabetes mellitus (T2DM) with peripheral arterial disease (PAD) patients was increased significantly (597.08) as compared to T2DM patients (231.34) and healthy individuals (16.93).